Low-invasive somatic oncogenes and lymph node metastasis in pediatric papillary thyroid cancer: implications for prophylactic central neck dissection

Objective The American Thyroid Association (ATA) Pediatric Guidelines recommend selective, prophylactic central neck dissection (pCND) for patients with papillary thyroid carcinoma (PTC) based on tumor focality, tumor size, and the surgeon’s experience. With the expansion of pre-surgical somatic oncogene testing and continued controversy over the benefits of pCND, oncogenic alteration data may provide an opportunity to stratify pCND. This study compared lymph node (LN) involvement in pediatric patients with PTC between tumors with low- and high-invasive-associated alterations to explore the potential utility of preoperative oncogenic alterations in the stratification of pCND. Methods This is retrospective cohort study of pediatric patients who underwent somatic oncogene testing post thyroidectomy for PTC between July 2003 and July 2022. Results Of 192 eligible PTC patients with postoperative somatic oncogene data, 19 tumors harbored somatic alterations associated with low-invasive disease (19/192, 10%), and 128 tumors harbored a BRAFV600E alteration (45/192, 23%) or an oncogenic fusion (83/192, 43%). Tumors with low-invasive alterations were less likely to present malignant preoperative cytology (2/18, 11%) than those with high-invasive alterations (97/124, 78%; P < 0.001). Twelve patients with low-invasive alterations had LNs dissected from the central neck (12/19, 63%) compared to 127 patients (127/128, 99%) with high-invasive alterations. LN metastasis was identified in two patients with low-invasive alterations (2/19, 11%) compared to 107 patients with high-invasive alterations (107/128, 84%; P < 0.001). Conclusion Pediatric patients with low-invasive somatic oncogenic alterations are at low risk for metastasis to central neck LNs. Our findings suggest that preoperative knowledge of somatic oncogene alterations provides objective data to stratify pediatric patients who may not benefit from pCND.


Introduction
Papillary thyroid carcinoma (PTC) is the most common pediatric endocrine malignancy.While long-term disease-specific survival approaches 100% for children and adolescents (1,2), recurrence following treatment may affect up to 30% of patients (3,4), with cervical lymph node metastasis (LNM) in central and lateral neck compartments being well-established sites of persistent and recurrent disease.The 2015 American Thyroid Association (ATA) Pediatric Guidelines recommend selective consideration of prophylactic central neck dissection (pCND) for patients with PTC based upon tumor focality, tumor size, and the experience of the surgeon (Recommendation 12B) (5).This recommendation was based on data demonstrating a high rate of metastasis (30-90%) (6, 7) to regional lymph nodes (LNs) in the pediatric population and was used to stratify patients into ATA risk levels for selective use of radioiodine therapy (RAIT; Recommendation 15) (5,8,9).In addition, the extent of initial LN resection has been shown to impact disease free survival (DFS) (1,5,10).However, it is important to consider the potential benefit of pCND against the increased risk of surgical complications (11,12).Based on this, the recent 2022 European Thyroid Association Guidelines for the management of pediatric thyroid nodules and differentiated thyroid carcinoma (DTC) recommended limiting pCND to patients with 'suspicious features of advanced thyroid cancer' (13).
Pre-operative assessment for surgical stratification commonly includes ultrasound (US) and fine needle aspiration (FNA), but these are highly subjective measures with wide variability in completeness and interpretation across institutions (14,15,16).Identifying a more objective preoperative marker that could predict the invasive behavior of the tumor, particularly tumors unlikely to metastasize to central neck LNs, would be clinically useful in decreasing reliance on pCND.If proven reliable, this marker would be associated with a high likelihood of surgical remission without placing patients at increased risk for potential surgical complications associated with pCND.
With the expansion of somatic oncogene analysis using next-generation sequencing (NGS), and data confirming associations between oncogenic alterations and invasive tumor behavior (17), knowledge of the somatic alteration preoperatively has the potential to further inform the surgical management of pediatric PTC (17,18,19,20).Oncogene test results are not subject to variability in provider interpretation and offer impartial foresight into the pathological subtype and metastatic behavior of a patient's disease (17,21).This study was undertaken to compare LN involvement in pediatric patients with PTC demonstrating low-and high-invasive associated alterations to assess the association between molecular subtype and N1 disease in an effort to determine if somatic oncogene testing could be used to guide pCND stratification.

Data collection
An IRB-approved study was conducted on patients with PTC <19 years old who underwent thyroid surgery and somatic oncogene testing at the Children's Hospital of Philadelphia (CHOP) between July 2003 and July 2022.Patient demographics, medical history, thyroid US, cytology, surgical approach, pathologic features, diagnosis, and postoperative somatic oncogene findings were extracted from the hospital electronic medical record system (Epic ® ).FNA cytology was classified according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) (22).A two-stage thyroidectomy was defined as a lobectomy followed by a completion thyroidectomy.Prophylactic CND (level VI or levels VI-VII) was performed for patients undergoing thyroidectomy without preoperative US evidence of central neck LN metastasis (American Joint Committee on Cancer (AJCC) N0a) and selected patients undergoing lobectomy with indeterminate (TBSRTC III-V) or malignant (TBSRTC VI) cytology.A therapeutic CND was performed for patients with preoperative evidence and FNA confirmation of central and lateral neck LN metastasis (AJCC N1b).
After surgery, primary tumor (T), regional LNs (N), and distant metastasis (M) were assigned using the 8th edition of the AJCC classification for PTC (23).Thyroid cancer risk stratification was adopted from the 2015 ATA Management Guidelines for Children with Thyroid Nodules and Differentiated Thyroid Carcinoma (5).Data analyses were limited to patients with a histological diagnosis of PTC.

Molecular genetic analyses
Sequential somatic oncogene testing was performed on all tumors based on a histological diagnosis of PTC without selection based on AJCC TNM.Differing somatic panels were used over the length of the study based on availability and changes in clinical practice.Somatic alterations were identified using the following targeted assays: (i) CHOP's Comprehensive Solid Tumor Panel (CSTP), (ii) Asuragen miRInform thyroid test, or (iii) Qiagen BRAF amplification test.CSTP comprises 238 genes and more than 600 fusions to detect single nucleotide variants, indels, gene fusions, and copy number alterations in pathologic specimens (24,25).The Asuragen miRInform thyroid test analyzes the presence of common variants in BRAF, HRAS, KRAS, and NRAS, and fusion transcripts in RET/PTC1, RET/PTC3, and PAX8-PPARγ in pathologic specimens, and does not analyze PTEN, DICER1, NTRK fusions, and novel RET fusions (26).Qiagen detects somatic mutations in the BRAF oncogene utilizing RT-PCR (27).

Statistical analysis
Genetic alterations were classified into two groups in accordance with previous studies demonstrating genotypic variations in behavior of pediatric PTC: lowinvasive alterations (DICER1, PTEN, RAS, and TSHR variants, BRAF non-V600E variants; and PAX8-PPARγ fusion) and high-invasive alterations (RET, NTRK, ALK, BRAF, or MET fusions and BRAF V600E variant) (17,18,28,29,30).Patients with germline mutations in thyroid cancer predisposition syndromes, such as PTEN Hamartoma Tumor Syndrome and DICER1 syndrome, were excluded from the analysis.Preoperative features, clinicopathologic characteristics, and outcomes were compared between patients with lowand high-invasive alterations.Categorical variables were summarized using frequency (percent) and were compared using the two-tailed Fisher exact test.Continuous variables were summarized using median (IQR = 1st-3rd quartiles) and were compared by Mann-Whitney U test (nonparametric).P-values ≤0.05 were considered statistically significant.All statistical analyses were performed in R 4.1.0and R Studio 1.4.1717 with packages tidyverse and rstatix (31,32,33).

Preoperative, cytologic, and histologic features
Clinicopathologic features of the 147 patients with somatic oncogenic alterations are presented in Table 2 and Fig. 1.Patients with low-invasive alterations were less likely to demonstrate lymphadenopathy  2).
Overall, 84% (107/128) of patients with high-invasive alterations demonstrated metastatic LNs (N1a/N1b), compared to 11% (2/19) of patients with low-invasive alterations (P < 0.001; Table 3).Of the 42 patients (42/128; 33%) with N1a disease in the high-invasive cohort, 29 (29/42; 69%) did not have pre-operative lymphadenopathy identified on ultrasound and/or physical exam.Only two patients with low-invasive alterations (BRAF T599del ; TSHR M453T ) had positive Clinicopathologic features of 19 patients with low-invasive and 128 patients with high-invasive somatic driver alterations who underwent thyroidectomy.Characteristics include age at the time of surgery, sex, preoperative lymphadenopathy, cytology (TBSRTC), histologic subtype, primary tumor (T) staging, regional lymph node (N) staging, distant metastasis (M) staging, ATA risk status, radioactive iodine (RAI) therapy, lymphatic invasion, and response to therapy at 1 year post initial treatment.Genetic alterations were categorized by driver.ATA, American Thyroid Association; CSTP, Comprehensive Solid Tumor Panel; PTC, Papillary Thyroid Carcinoma; TBSRTC, The Bethesda System for Reporting Thyroid Cytopathology. .By contrast, mild diffuse activity in the lungs was identified on a post-radiotherapy scan for one patient with a low-invasive NRAS Q61R variant.Pathological examination revealed an encapsulated, multifocal, and bilateral fvPTC with lymphatic and vascular invasion, which was subsequently staged as T2N0bM1.This patient did not have tissue available to assess for a combined RAS-EIF1AX variant, which may be associated with more invasive disease in adult patients (36).
Forty-six patients (46/128; 36%) had a pCND in the high-invasive cohort.Of these patients, 61% (28/46) had LNM and no pre-operative evidence of central neck LN disease on pre-operative US, demonstrating oncogene analysis adds information about the metastatic potential that US and physical examination may miss.Furthermore, in nine patients with indeterminate cytology (Bethesda III or IV) and detection of a high-invasive oncogene, five patients (5/9, 56%) were found to have LNM -60% (3/5) with N1a disease found on pCND and 40% (2/5) with N1b disease.Thus, pre-operative identification of an oncogene may be used to stratify which patients with indeterminate cytology may benefit from pCND.Of the 11 patients with pCND in the low-invasive cohort, 91% (10/11) were found to have no LN metastasis (N0a disease), showing there is potential to stratify patients out of the pCND category using somatic oncogene analysis.
Our observations are aligned with previous pediatric (17,37,38) and adult (39,40,41) studies evaluating the applicability of molecular oncogenic alteration classification to predict invasive behavior of PTC.While the Thyroid Cancer Genome Atlas (21) and other reports (39) have grouped PTC into RAS-like and BRAFlike oncogenic driver groups for low-and high-invasive behavior in adult PTC, pediatric-specific somatic oncogene analysis suggests that a three-tiered system more accurately describes increasing risk of invasive behavior: (i) RAS-like oncogenes (PTEN, DICER1, RAS, and PAX8-PPARγ) are associated with a low risk for LNM; (ii) BRAF V600E is associated with a high risk for regional (N1) LNM; and (iii) fusion oncogenes (RET, NTRK, ALK, BRAF, or MET fusions) are associated with a high risk for regional (N1) and distant (M1) metastasis (17,21,39).
The results of our study hold potential clinical applicability as discussion remains over the benefit of pCND in the initial approach to management of pediatric patients with PTC.The 2015 ATA Pediatric Guidelines (5), as well as several recent publications (42,43), rely on N status to stratify the extent of initial surgery (43) and RAIT (5,42).There are, however, concerns over increased surgical complications associated with pCND (12), with the recent European Thyroid Association Guidelines for the management of pediatric thyroid nodules and differentiated thyroid carcinoma recommending to limit pCND to patients with 'suspicious features of advanced thyroid cancer' (13).Preoperative cytopathology may aid in patient selection, limiting pCND to patients with TBSRTC V and VI cytology (20).However, the invasive behavior of PTC, including sonographic assessment of LN disease and cytological classification, is subjective a One patient with a low-invasive PAX8-PPARγ fusion underwent a CND and selective removal of two LNs.Operative and pathology records report no metastatic LNs, however the total number of LNs removed was not reported and hence excluded from the quantitative analysis.b pCND was indicated for patients having (i) no lymphadenopathy on preoperative ultrasound and/or physical examination by provider and/or (ii) FNA confirmation of benign LNs.Therapeutic LN dissection was indicated for patients having (i) central neck lymphadenopathy on preoperative ultrasound with FNA confirmation of malignancy or (ii) lateral neck lymphadenopathy on preoperative ultrasound with FNA confirmation of malignancy or (iii) preoperative ultrasound demonstrating rounded, echogenic LNs with peripheral blood flow along with an invasive-appearing lesion inside the thyroid with FNA confirmation of being suspicious for malignancy or malignant.IQR, interquartile range (25th-75th percentile); LN, lymph node, pCND, prophylactic central neck dissection.
with wide variation across institutions limiting the ability to make generalized recommendations based solely on US and cytology (15).The incorporation of somatic oncogene testing into the preoperative assessment may afford an opportunity to limit pCND for patients with nodules harboring low-invasive somatic alterations that demonstrate low-risk features on preoperative ultrasound (i.e.smooth margins and wider-than-tall shape and no evidence of extrathyroidal extension, punctate echogenic foci, or regional adenopathy (N0b status)) and TBSRTC indeterminate cytology (categories III/atypia of undetermined significance and IV/follicular neoplasm) (20).
The limitations of this study are its single-center retrospective design, variance in somatic oncogenic panels, a lower percentage of patients with low-invasive alterations undergoing CND compared to patients with high-invasive alterations, and lack of somatic oncogene data for all tumors.Despite these limitations, the data on the lower rate of central neck LNM in tumors with low-invasive somatic oncogenic alterations are intriguing (Table 4) and support the potential benefit of prospective studies exploring the utility of preoperative molecular testing to stratify surgical approach to care.
Current efforts are underway to increase the sample size and perform multi-omics analysis to further delineate the genomic landscape of pediatric PTC beyond somatic oncogene driver.One of the goals of this initiative is to identify additional markers of invasive behavior for the 16% (21/128) of tumors that were found to have a high-invasive somatic alteration but did not have metastasis to the central neck compartment (N0; Table 3).Prospective, multicenter studies are warranted to confirm whether the incorporation of all available data, including ultrasonography, cytology, and somatic oncogene, can be incorporated into clinical practice in an effort to reduce potential surgical complications of pCND without compromising the ability to stratify RAIT based on current pediatric risk stratification systems or to achieve remission from disease (5,42).To this end, the authors have helped create the Child and Adolescent Thyroid Consortium (CATC), an international pediatric thyroid consortium designed to enhance collaboration between multidisciplinary pediatric thyroid centers (www.thyroidcatc.org).

Conclusion
Pediatric patients with low-invasive somatic oncogenic alterations are at low-risk for metastasis to central neck LNs.Our findings suggest that preoperative knowledge of somatic oncogene alterations provides objective data to stratify pediatric patients who may not benefit from pCND.Future prospective studies are needed to validate if comprehensive somatic NGS panels for pediatric patients with indeterminate cytology can be used to optimize surgical management, limiting pCND to tumors with high-invasive somatic variants and fusions and not performing pCND for tumors with low-invasive alterations.

Table 1
Demographics and medical history of pediatric patients with papillary thyroid carcinoma and somatic driver alterations confirmed by postoperative oncogene testing.Data are presented as n (%).

Table 2
Clinicopathologic characteristics of pediatric patients with papillary thyroid carcinoma stratified by risk of driver alteration.Data are presented as n (%).

= 147 Driver alteration P Low-invasive, n = 19 High-invasive, n = 128
a Preoperative lymphadenopathy was determined through a review of thyroid ultrasound images and/or physical examination findings; b FNA was classified according to the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC).The highest category on primary tumor and/or lymph node(s) is reported.The proportion of malignant cytology was compared with two-tailed Fisher exact test; c Values are median (IQR).AUS, atypia of undetermined significance; FNA, fine needle aspiration; IQR, interquartile range (25th-75th percentile); PTC, papillary thyroid carcinoma.alterations.Twelve patients (12/19, 63%) with lowinvasive alterations had LNs dissected from the central neck compartment compared to 127 patients (127/128, 99%)

Table 3
Postoperative characteristics of pediatric patients with papillary thyroid carcinoma stratified by risk of driver alteration.Data are presented as n (%).TSHR M453T .Evaluation of LNs was limited preoperatively for the patient with BRAF T599del , the only patient in the low-invasive cohort found to have N1a disease despite negative preoperative US.

Total, n = 147 Driver alteration P Low-invasive, n = 19 High-invasive, n = 128
TNM was adopted from the AJCC 8th Edition Staging and evaluated within 12 months post-initial surgery; b Values are median (IQR); c Thyroid cancer risk stratification was adopted from the '2015 ATA Management Guidelines for Children with Thyroid Nodules and Differentiated Thyroid Cancer' and evaluated at initial surgery.Extensive involvement was defined as >5 positive lymph nodes.ATA, American Thyroid Association; AJCC, American Joint Committee on Cancer; IQR, interquartile range (25th-75th percentile); RAIT, radioactive iodine therapy; TNM, tumor-node-metastasis. a

Table 4
Lymph node involvement in pediatric patients with papillary thyroid carcinoma stratified by risk of diver alteration.Data are presented as n (%) or as median (IQR).